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Abstract:

An isolated or purified compound is provided, comprising
A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc.
There is further provided a vaccine based on such compound, having
particular use to treat or prevent an infection caused by a Campylobacter
organism. There is also provided an antibody or antisera against the
compound, having particular use to diagnose the presence of an infection
caused by a Campylobacter organism.

Claims:

1. An isolated or purified compound comprising
A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc.

2. The compound of claim 1 connected or linked to a single amino acid, an
oligopeptide, a peptide, a protein, or a lipid.

3. The compound of claim 2 wherein said single amino acid is asparagine.

4. A vaccine comprising the compound of claim 1.

5. The vaccine of claim 4, further comprising an adjuvant.

6. (canceled)

7. (canceled)

8. A method of treating or preventing an infection caused by a
Campylobacter organism, comprising administering the vaccine of claim 4
to an animal or human in need thereof, wherein the compound comprises a
glycan that is native to said organism.

10. A method of improving the productivity and health of an animal herd
or health of a human by administering to said herd or said human the
vaccine of claim 4.

11. A method of producing an antibody or antiserum comprising the steps
of inoculating an animal or a human with the compound of claim 1 to
stimulate an immune response to said compound in said animal or said
human, withdrawing serum from said animal and optionally purifying said
serum to obtain the antibody or purified antiserum.

12. An antibody or antiserum effective against a Campylobacter organism,
wherein said antibody or antiserum is prepared by the method of claim 11,
and wherein the compound of claim 1 is native to said organism.

14. A method of diagnosing the presence of an infection in an animal or a
human caused by a Campylobacter organism, comprising obtaining a sample
from the animal or the human, and contacting the sample with the antibody
or antiserum of claim 12, wherein detection of said organism by the
antibody or antiserum indicates the presence of the infection.

Description:

FIELD OF THE INVENTION

[0001] The invention relates to an N-linked glycan compound of Formula 1,
which optionally may be fused or attached to an amino acid, peptide,
protein or lipid. The invention further relates to antibodies and
antisera against such compound, and the use thereof to diagnose an
infection caused by a Campylobacter pathogen. The invention further
relates to the use of the compound as a vaccine to treat or prevent
infection by a Campylobacter pathogen.

BACKGROUND

[0002] Campylobacter jejuni and Campylobacter coli are the two most
commonly isolated species of campylobacter that cause human infection.
These organisms cause high rates of gastroenteritis worldwide, with the
number of cases often exceeding that for Salmonella, Shigella and
Enterotoxigenic E. coli combined (Butzler J P, Clinical Microbiology and
Infection 2004). Furthermore, C. jejuni infection has been linked to the
development of Guillain-Barre Syndrome, the most common cause of
pathogen-caused paralysis since the eradication of polio (for reviews
see: Kaida K, Glycobiology, 2009; Bereswill S & Kist M, Current Opinion
in Infectious Diseases, 2003). Other Campylobacter species have been
recognized as emerging pathogens in human gastroenteritis (C.
upsaliensis, C. hyointestinalis), were associated with inflammatory bowel
disease in children, with gingivitis, periodontitis and human abortions
(C. retus, C. concisus) (Zhang L S et al., Journal of Clinical
Microbiology, 2009) and in causing venereal disease and infertility in
livestock (especially cattle; C. fetus venereal's), and sheep abortions
(C. fetus fetus) (Butzler J P, Clinical Microbiology and Infection, 2004
and references therein).

[0004] In eukaryotes, glycosylated proteins are ubiquitous components of
extracellular matrices and cellular surfaces. Their oligosaccharide
moieties are implicated in a wide range of cell-cell and cell-matrix
recognition events that are vital in biological processes ranging from
immune recognition to cancer development. Glycosylation was previously
considered to be restricted to eukaryotes, however through advances in
analytical methods and genome sequencing, there have been increasing
reports of both O-linked and N-linked protein glycosylation pathways in
bacteria (Nothaft H & Szymanski C M, Nature Reviews Microbiology, 2010).
Since the discovery of the first general protein glycosylation pathway in
bacteria (Szymanski C M et al., Molecular Microbiology 1999), the
demonstration that the C. jejuni glycans are attached through an
N-linkage en bloc (Kelly J H et al., Journal of Bacteriology 2006, Wacker
M et al., Science 2002, Young N M et al., Journal of Biological
Chemistry, 2002) and that the pathway not only can be functionally
transferred into Escherichia coli (Wacker M et al., Science, 2002), but
that the oligosaccharyltransferase enzyme (PgIB) is capable of adding
foreign sugars to protein (Feldman M et al., PNAS 2005), a surge of
research activities has resulted in further characterization and
exploitation of this system.

[0005] The detailed structure of the unique C. jejuni N-linked
heptasaccharide has been described (Young N M et al., Journal of
Biological Chemistry, 2002). Using methods such as high resolution magic
angle spinning (HR-MAS) NMR (Szymanski C M et al., Journal of Biological
Chemistry, 2003), it has been shown that this heptasaccharide is
conserved in structure in both C. jejuni and C. coli.

[0006] An intermediate in the C. jejuni N-linked glycosylation pathway has
been described, namely a free (oligo-) heptasaccharide (fOS)--a soluble
component of the C. jejuni periplasmic space (Liu X et al., Analytical
Chemistry, 2006). This fOS has the identical structure as the N-linked
oligosaccharide added onto proteins (Nothaft H et al., PNAS 2009). Under
laboratory growth conditions, the ratio of fOS versus heptasaccharide
N-linked to protein is approximately 9:1. The fOS in C. jejuni plays a
role in osmoregulation similar to bacterial periplasmic glucans and this
pathway can be manipulated by altering the environmental osmolyte
concentration (Nothaft H et al., PNAS 2009).

[0008] We have determined the N-glycan and fOS structures from a number of
Campylobacter species, all of which possess N-linked glycans and fOS. In
addition, we demonstrated that campylobacter N-glycans and fOS can be
divided into two structural groups. The first group produces a similar
structure to that published for C. jejuni and C. coli (Young N M et al.,
Journal of Biological Chemistry, 2002; Szymanski C M et al., Journal of
Biological Chemistry, 2003). The second group produces a unique glycan
structure which differs from that determined for C. jejuni and C. coli
and that have never been described before. Campylobacter species that
fall into this group include Campylobacter fetus venerealis (cause of
venereal disease and infertility in cattle), Campylobacter fetus fetus
(cause of sheep abortions), Campylobacter concisus (associated with
gingivitis and periodontitis, and has been isolated from the feces of
patients with gastroenteritis), Campylobacter hyointestinalis (like C.
jejuni and C. coli, is associated with diarrheal disease) and
Campylobacter hyointestinalis subspecies.

[0010]FIG. 2 illustrates a phylogenetic analysis of the protein sequences
of the key component of this pathway, the oligosaccharyltransferase
(PgIB) including the genome sequenced Campylobacter species and other
related organisms and demonstrates that the Campylobacters divide into
two groups. Within the campylobacter branch Structure 1 producing species
are in the upper box, Formula 1A and Formula 1B producing strains are in
the lower box (adapted from Nothaft H & Szymanski C M, Nature Reviews
Microbiology, 2010).

[0012] According to one aspect, the invention relates to a novel N-linked
glycan (referred to as N-glycan) compound of Formula 1:
A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc.
This compound in its native form is common to several Campylobacter
species. In its native form, the compound is soluble in the periplasm as
well as attached to inner membrane and periplasmic proteins and most
notably surface outer membrane proteins of many Campylobacter species,
including pathogens. In the present invention, the compound of Formula 1
is provided in isolated and/or purified form. The compound comprises two
hexasaccharides which differ from each other in a terminal sugar, which
comprises either Glc or GlcNAc. The first of said compounds is:
GlcNAc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc (herein Formula 1A). The
second of said compounds is: Glc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc
(herein Formula 1B).

[0013] In the above Formula 1, QuiNAc4NAc represents an alternative
signifier of the saccharide Bac, which constitutes an abbreviation of
bacillosamine.

[0014] In one aspect the invention relates to an isolated or purified
compound comprising the compounds of Formula 1 connected or linked to a
single amino acid, an oligopeptide, a peptide, a protein, or a lipid. In
one aspect, the oligopeptide or peptide comprises between 2 and 40 amino
acids, or between 2 and 30 amino acids, or between 2 and 20 amino acids,
or between 2 and 10 amino acids.

[0015] The invention further relates to a method of producing an antibody
or antiserum comprising the steps of providing the compound of Formula 1,
inoculating an animal or humans with said compound to stimulate an immune
response to said compound, withdrawing serum from said animal and
optionally purifying said serum to obtain the antibody or antiserum. The
resulting antibody or antiserum binds to Campylobacter species wherein
the glycan described herein is native thereto, including Campylobacter
fetus venerealis, Campylobacter fetus fetus, Campylobacter concisus,
Campylobacter hyointestinalis and Campylobacter hyointestinalis
subspecies, Campylobacter sputorum and Campylobacter sputorum subspecies,
Campylobacter Ianienae, Campylobacter ureolyticus, Campylobacter hominis,
Campylobacter gracilis, Campylobacter rectus, Campylobacter showae,
Campylobacter mucosalis and Campylobacter curvus.

[0016] The antibody or antiserum can be used for diagnostic purposes, to
detect the presence of said organisms in an animal or in a human.

[0017] Compounds of the present invention may be used in a vaccine
formulation, with or without an adjuvant, against Campylobacter fetus
venerealis, which is a major cause of reproductive failure in cattle and
for which the current vaccine is of limited use, or against other
Campylobacter species wherein the glycan of Formula 1 is native to such
organism, including the species listed above. Compounds of the present
invention have possible uses in protein glycoprotein engineering,
therapeutic and diagnostic applications. The invention thus relates to a
vaccine comprising the compound of Formula 1, optionally connected or
linked to a single amino acid, an oligopeptide, a peptide, a protein, or
a lipid. The single amino acid may comprise asparagine.

[0018] The invention further relates to the use of said vaccine to treat
or prevent an infection caused by a Campylobacter organism, wherein the
compound of Formula 1 comprises a native glycan within said organism, and
a method of treatment comprising said use, within a human or animal.

[0019] According to another aspect, the invention relates to a method of
improving the productivity and health of an animal herd by administering
to said herd the vaccine as described above.

[0020] The vaccines, antibodies and antisera described herein may also be
used to for prevention, treatment and diagnosis in humans.

[0033] The present invention relates to the glycan compound
A-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc, wherein A is GlcNAc or Glc.
The above compound encompasses the two glycan compounds
GlcNAc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc (herein Formula 1A) and
Glc-GlcNAc[GlcNAc]-GalNAc-GalNAc-QuiNAc4NAc (herein Formula 1B).

[0034] In the above Formulae, QuiNAc4NAc represents an alternative
signifier of the saccharide Bac, which constitutes an abbreviation of
bacillosamine (also known as diNAcBac). The compound of Formula 1 is
optionally connected or linked to a single amino acid, an oligopeptide, a
peptide, a protein, or a lipid.

[0035] Said lipid can be isolated and purified from a bacterial, archaeal
or eukaryotic source or can be chemically synthesized. Said linkage of
the glycan compound to the lipid can be mediated by a phosphate, a
pyrophosphate linker or by a glycosidic linkage. Examples of lipids (with
various chain lengths, saturation grade and configuration) linked to
N-glycans were described (Faridmoayer et al., Journal of Biological
Chemistry, 2009; Chen M M et al., Biochemistry, 2007). Lipid-linked
N-glycan compounds produced in the native host or in a heterologous
expression system include undecaprenyl-phosphate-linked N-glycan
compounds as shown for the C. jejuni N-glycan (Reid C W et al.,
Analytical Chemistry, 2008, Reid C W et al., Analytical Chemistry, 2009)
and proposed for the C. lari N-glycan (Schwarz F et al., Glycobiology
2011)) and N-glycan-LipidA conjugates (shown for the N-glycan of C.
jejuni (van Sorge N M et al., Cellular Microbiology, 2009)).

[0036] It has been determined that the above compound is substantially
conserved across multiple species of Campylobacter.

[0037] FIGS. 3A-3D depict N-glycans and fOS in select Campylobacter
species. (A) Western Blot using antiserum that recognizes the N-linked
hepta-saccharide of C. jejuni cross-reacted with other Campylobacter
species (open boxes) that also reacted with (B) soybean agglutinin
recognizing terminal GalNAc residues, but shows little reactivity with
(C) wheat-germ agglutinin (WGA) that recognizes terminal GlcNAc residues
present in Formula 1A and Formula 1B. Species that did not react with the
C. jejuni-specific antiserum but reacted with WGA were highlighted. (D)
Examples of mass spectrometry of fractions enriched for fOS or Asn-linked
of (1) C. jejuni (2) C. fetus venereals, (3) C. concisus, (4) C. fetus
fetus, and (5) C. hyointestinalis; results of all species analyzed by
mass spectrometry are summarized in Table 1.

[0038] Campylobacter jejuni 11168, C. concisus, C. hyointestinalis, C.
fetus fetus and C. fetus venerealis were grown under microaerobic
conditions. Whole cells obtained after centrifugation were digested with
large excess of proteinase K at pH 8 (adjusted by addition of ammonia) at
37° C. for 48 hours. Products of digestion or free
oligosaccharides were separated on Sephadex G-15 column (1.5×60 cm)
and each fraction eluted before the salt peak was dried and analyzed by
1H NMR. Fractions containing desired products were separated by
anion exchange chromatography on a Hitrap Q column (5 mL size, Amersham)
and the glycans were eluted with a linear gradient of NaCl--(0-1 M, 1 h)
that resulted in the isolation of a mixture of both glycan compounds
(Formula 1A and Formula 1B). Desalting was performed on Sephadex G15
prior to analysis by NMR.

[0040]FIG. 4 A is the 1H NMR spectrum of purified fOS from C. fetus
fetus. FIG. 4B overlay of 2D HSQC spectra for C. fetus fetus and C. fetus
venerealis indicating that fOS structures from both species are
identical. The NMR spectrum can also be overlaid with one obtained for C.
concisus (not shown). The corresponding chemical shifts δ (ppm) for
the purified free oligosaccharide from C. fetus fetus (as shown in FIG.
4A) are summarized in Table 2. Carbon and proton chemical shifts were
referenced to an internal acetone standard (δH 2.225 ppm, δC
31.07 ppm).

[0041] The campylobacter glycans that are either added to protein or
appear in a free form (FOS) can be divided into two structural groups.
The first group of Campylobacter species produces a unique glycan
structure that was previously determined for C. jejuni and C. coli and
herein for C. upsaliensis. Campylobacters which fall into the second
group consist of Campylobacter fetus venerealis (cause of venereal
disease and infertility in cattle), Campylobacter fetus fetus (cause of
sheep abortions), Campylobacter concisus Campylobacter hyointestinalis,
Campylobacter hyointestinalis subspecies, Campylobacter sputorum and
Campylobacter sputorum subspecies, Campylobacter lanienae, Campylobacter
ureolyticus, Campylobacter hominis, Campylobacter gracilis, Campylobacter
rectus, Campylobacter showae, Campylobacter mucosalis and Campylobacter
curvus.

[0042] Structure determination by NMR using large scale purified free
oligosaccharides (fOS) from C. fetus fetus, C. fetus venerealis, and C.
concisus demonstrated that this second group of campylobacters produced a
structure different from that originally described for C. jejuni and C.
coli (FIG. 4 and FIG. 5).

Preparation of Glycan of Formula 1 Compounds Linked to Single Amino Acid

[0043] A Pronase E digest of whole cell extracts obtained after lysis of
intact cells followed by mass spectrometry as described by Liu X. et al.,
AnalChem, 2005 and Nothaft H. et al., Methods Mol Biol, 2010 identified
the C. jejuni heptasaccharide (structure 1) attached to a single
asparagine and Formula 1A linked to a single asparagine in C. fetus fetus
(Table 1).

Example 4

Expression of Formula 1 Compounds

[0044] The protein glycosylation operon encoding all the genes necessary
for the production and transfer of Formula 1A and Formula 1B compounds
can be cloned and expressed from an E. coli plasmid(s). Alternatively,
the glycosyltransferases on a plasmid described by Wacker et al, Science
2002 that contains the C. jejuni protein glycosylation (pgl) operon can
be exchanged by Formula 1A and Formula 1B producing glycosyltransferases.
Expression of Formula 1A and Formula 1B compounds can be done in a
heterologous system in the presence of an affinity-tagged acceptor
peptide for N-linked protein glycosylation (already shown for the C.
jejuni N-glycan and for the C. lari N-glycan Wacker et al., Science 2002,
Schwarz et al., Glycobiology 2011) or as a fusion of such a protein with
a phage protein (Duerr et al., Glycobiology, 2010). The glycan containing
protein/peptide/phage can be purified by affinity-tag purification, if
necessary in combination with lectin or glycan-recognizing agent affinity
chromatography to separate the glycosylated and the non-glycosylated
peptides.

[0048] New Zealand White Rabbits were immunized with 2 mg of each of the
glyco-conjugate compounds prepared in Example 6, using a 6 week
immunization protocol (approved Animal Care Committee protocol No. 717).
After an initial subcutaneous injection (at 3 sites, 0.5 ml was injected
at each site) of 2.0 mg antigen using Freund's complete adjuvant (in a
1:1 ratio with the antigen), a booster dose with 2.0 mg mg of each
Formula 1A-BSA and Formula 1B-BSA conjugates mixed with Freund's
incomplete adjuvant (in a 1:1 ratio with the antigen) was given
subcutaneously (at 3 sites 0.5 ml were injected at each site) after 4
weeks. After 6 weeks serum from a 5 ml blood sample from each animal was
prepared by cooling the blood sample for 60 min on ice followed by
centrifugation for 20 min at 10.000×g. Individual sera were
analyzed for the production of Formula 1A and Formula 1B-specific
antibodies by Western Blotting with Campylobacter whole cell lysates
(FIG. 8).

[0049]FIG. 8 shows an immuno-blot with antiserum that was raised against
the single BSA-glycoconjugates: 120 μg of whole cell lysates from
either C. jejuni 11168 wild-type (lane 1), C. jejuni 11168 pgIB mutant
strain (lane 2) or C. fetus fetus (lane 3) were applied to 12.5%
SDS-PAGE. After transfer to a PVDF membrane, the immobilized proteins
were probed (A) with a 1:2000 dilution of a serum sample obtained from a
rabbit that was immunized with BSA-Formula 1B compound (SZR-1) and with
(B) serum of a rabbit immunized with BSA-Formula 1A compound (SZR-3).
Molecular weight markers (MW in KDa) are indicated on the left.

[0051] Glycopeptides were isolated and identified from Campylobacter fetus
fetus, Campylobacter fetus venerealis, and Campylobacter concisus with
the results shown in Table 3. The glycan portions there of all comprised
the compound of Formula 1A or 1B.

[0052] List of proteins identified to be N-linked with Formula 1A and
Formula 1B. The single peak areas for Formula 1A and Formula 1B were
determined by multiple reaction monitoring (MRM) mass spectrometry.

[0053] FIGS. 9A-F depict MS spectra showing that both Formula 1A and
Formula 1B compounds are N-linked (to the same peptide), as follows:

[0057]FIG. 11 shows immunoblots with antiserum that was raised either
against Formula 1A or Formula 1B or with and antiserum that targets the
N-glycan of C. jejuni (structure 1, hR6 was described by Schwarz et al.,
Nature Chemical Biology, 2010). 90 μg of C. fetus fetus (lane 1), C.
fetus venerealis (lane 2), C. concisus (lane 3), C. hyointestinalis (lane
4) and C. jejuni 11168 (lane 5) were applied to 12.5% SDS-PAGE. After
transfer to a PVDF membrane the immobilized proteins were probed with (A)
a 1:500 dilution of a serum sample obtained from a rabbit that was
immunized with BSA-Formula 1B compound (SZR-1), with (B) a 1:500 dilution
serum of a rabbit immunized with BSA-Formula 1A compound (SZR-3) or (C)
with a 1:5,000 of an antiserum specific against the N-glycan of C. jejuni
(hR6). Molecular weight markers (MW in KDa) are indicated on the left.

[0058] The glycan compounds (Formula 1A and Formula 1B) can be attached to
various glycan carriers (peptides, lipids). The resulting compounds can
be used to stimulate an immune-response against the respective structure
that will be protective against infection with Formula 1A and Formula 1B
presenting bacterial species.

[0059] Generated antisera/antibodies can be used (when i.e immobilized on
a surface) as a diagnostic to detect e.g. C. fetus venerealis or C. fetus
fetus in infected livestock (especially C. fetus venerealis cattle) or to
detect human pathogenic Campylobacter strains (e.g. C. concisus, C.
hyointestinalis, C. ureolyticus). Said antisera/antibodies can be used to
detect compounds in any body fluid or secretion. For example, bull semen
could be tested with antibodies recognizing the glycan of Formula 1 to
detect Campylobacter fetus venerealis infection that may be present in
the animal.

[0060] The compounds of the present invention can be used to immunize
animals, in particular livestock, against C. fetus venerealis, C. fetus
fetus, and other Campylobacter species in which the glycan described
herein is native to the organism. Immunization can take the form of
treating or preventing disease in individual animals or on a herd-wide
basis for improved productivity and health of the herd.

[0061] To the extent that Campylobacter species in which the glycan of
Formula 1 is native to the organism, the compounds described herein can
be used in a similar fashion to the above for preparing vaccines to treat
or prevent infection by such organisms within humans. As well, a similar
diagnostic function can be obtained in humans, using the antibodies or
antisera raised against such compounds. Similarly, the compounds can be
targeted by other therapeutics such as bacteriophages or their receptor
binding proteins.

[0062] The present invention has been described by way of various
embodiments thereof. It will be understood by persons skilled in the art
that the invention is not limited in scope to such embodiments. Rather,
the full scope of the invention encompasses and may be appreciated by
reference to this patent specification in its entirety, including the
claims thereof, and including modifications, variations, and alternative
embodiments that would be understood to the skilled person based on said
specification.